Combinatorial chemistry has developed into a useful method for the rapid synthesis of new compounds for drug discovery (see Lam, K. S. et al. Nature 1991, 354, 82-84). However, a crucial step in this drug discovery process is the development of high-throughput in vitro enzyme assays to identify positive hits from such combinatorial libraries.
For example, library screening has identified small molecule sulfotransferase inhibitors (see Armstrong, J. I. et al. Angew. Chem. Int. Ed. 2000, 39, 1303-1306). Meldal has reported using fluorescent resonance energy transfer (FRET) substrates and one-bead-two-compounds library approaches to screen protease inhibitors (see Meldal, M. Biopolymers 2002, 66, 93-100). A potent and specific sialyltransferase inhibitor, Soyasaponin I, has been discovered by screening over 7500 microbial extracts and natural products (see Wu, C. Y. et al. Biochem. Biophys. Res. Commun. 2001, 284(2), 466-469). More recently, a peptide WWWWNG-NH2 was identified as a potent inhibitor for α2,3-sialyltransferase (ST3Gal I) from the screening of a combinatorial peptide library (see Lee, K. Y. et al. J. Biol. Chem. 2002, 277, 49341-49351).
Another pharmaceutically important enzyme is neuraminidase (NA). Neuraminidase is an enzyme that cleaves the α-ketosidic linkage of the terminal sialic acids and has been found in viruses, bacteria, parasites, and mammalian cells (see Corfield, T. Glycobiology 1992, 2, 509-521; and Corfield, A. P. et al. “Role of sialidases and sialic acids in molecular recognition phenomena” in conferences Philippe Laudat 1991 pp. 113-134, Institute National de la Sante et de la Racherche Medicale, (INSERM), Paris; Rosenberg and Schengrund Biological roles of sialic acids Plennum Press, N.Y, 1976, pp. 295-360). It plays an important biological role in the regulation of glycoconjugates involved in cell-to-cell interactions (see Schauer, R. Adv. Carbohydr. Chem. Biochem. 1982, 40, 131-134). For example, influenza viruses have two surface glycoproteins, hemaggutinin and neuraminidase. Hemagglutinin binds to receptors containing neuraminic acid, which allows the virus to penetrate through the cell membrane. Neuraminidase destroys receptors recognized by hemaggutinin by cleaving the α-ketosidic linkage of sialic acids. This cleavage facilitates passage of the virus to and from sites in the respiratory tract (see Colman, P. M. “Neuraminidase: enzyme and antigen” in The Influenza Viruses (Klug, R. M., ed.), 1989, pp. 175-218. Plenum Publishing Corporation, New York). Studies with a neuraminidase-deficient influenza virus have shown that the mutant virus is still infective but the budding virus particles form aggregates or remain bound to the infected cell surface (see Liu, C. et al. J. Virol. 1995, 69, 1099-1106). Influenza causes considerable disease burden each year and while vaccination is the first line of defense against influenza A and B viruses, antiviral therapy can aid in controlling the impact of the disease (see Schmidt Drugs 62: 2031 (2004)).
This important biological activity has prompted chemists to use the crystal structure of neuraminidase to design specific inhibitors as anti-influenza virus agents (see FIG. 1 from von Itzstein, M.; et al. Nature, 1993, 363, 418-423; and Taylhell, N. R. et al. J. Med. Chem. 1998, 41, 798-807; and FIG. 2. from Crennell, S. J. et al. J. Mol. Biol. 1996, 259, 264; and Structure 1994, 2, 535-544 (see also Kim, C. U. et al. J. Am. Chem. Soc. 1997, 119, 681-690; Babu, Y. S. et al. J. Med. Chem. 2000, 43, 3482-3486; Chand, P.; et al. J. Med. Chem. 2001, 44, 4379-4392; Andrews, D. M. et al. Eur. J. Med. Chem. 1999, 34, 563-574; and Kiefel, M. J. and von Itzstein, M. Chem. Rev. 2002, 102, 471-490). Since the determination of the crystal structure of the influenza neuraminidase, (see Varghese, J. N. et al. Nature 1983, 303, 35-40; Varghese, J. N. and Colman, P. M. J. Molec. Biol. 1991, 221, 473-486; Burmeister, W. P. et al. EMBO J. 1992, 11, 49-56; and Varghese, J. N. et al. Protein Science 1995, 4, 1081-1087) many derivatives of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) have been designed as transition state analogues to block the catalytic sites of influenza neuraminidase (see Varghese, J. N. Drug Development Res. 1999, 46, 179-196, Palese and Schulman in Chemoprophylaxis and Virus Infections of the Upper Respiratory Tract vol. 1 CRC Press Cleveland (Oxfod, J. S. ed.) 1977 pp. 189-205; and references cited therein). This has lead to the development of highly potent influenza neuraminidase inhibitors ZANAMIVIR™ and OSELTAMIVIR™, which are currently in use for treatment of influenza virus infection (see Gubareva, L. V. et al. Lancet 2000, 355, 827-835; Holzer et al. Glycoconj. J. 10: 40-44 (1993); Chong et al. Biochem. Int. 24: 165-171 (1991); and references cited therein). More recently, the synthesis and biological evaluation of a functionalized cyclopentane analog, RWJ-270201, has been reported (Chand et al. J. Med. Chem. 44: 4379 (2001). This compound is a potent inhibitor of wild-type NA and some ZANAMIVIR™- and OSELTAMIVIR™-resistant influenza A and B virus variants (Gubareva, L. V. et al. Antimic. Agent and Chemother. 2001, 45, 3403-3408).
Success in achieving selective NA inhibition by modifying the glycerol side chain to increase hydrophobic interactions led to several other studies of ZANAMIVIR™ analogs. Honda et al. published a series of papers describing the synthesis and biological evaluation of 7-O alkylated ZANAMIVIR™ analogs. Their first studies showed that the 7-hydroxyl could be replaced with fluorine to give an improved activity profile (see above). Methylation of the 7-hydroxyl led to slightly diminished anti-NA activity but the ethyl ether was actually more active than ZANAMIVIR™ in the NA assay and both ethers showed increased activity in plaque reduction assays (see Honda et al. Biorg. Med. Chem. Lett. 12: 1921-24 (2002); Honda et al. Bioorg. Med. Chem. Lett. 12: 1925-28 (2002)). In general, compounds with alkyl ethers of less than 12 carbons showed potent (nM) inhibition of NA and improved activity relative to ZANAMIVIR™ in cell-culture assays. These combined studies indicate that modifications of the glycerol side-chain of ZANAMIVIR™ are tolerated and often beneficial.
Solution-phase neuraminidase inhibition assays normally use the fluorogenic substrate, 2′-(4-methylumbelliferyl)-α-D-acetyl-neuraminic acid, which is cleaved by neuraminidase to yield a fluorescent product that can be quantified using a fluorometer (Potier et al., Anal. Biochem. 94:287-296 (1979)), however, this assay method is not amenable to a high-throughput format. In addition, due to the fast emergence of resistant viral strains (see McKimm-Breschkin, J. L. Antiviral Res. 2000, 47, 1-17), there remains a need to find new influenza neuraminidase inhibitors.
Another pharmaceutically important class of enzymes are sialyl transferases. Glycoconjugates, including glycoproteins, glycosphingolipids, and polysaccharides, play important biological roles in cell-cell recognition, bacterial adhesion, signal transduction, and immune response (see Fukuda, M. “Roles cell surface carbohydrate” in Molecular and Cellular Glycobiology (Fukuda and Hindsgaul, eds.); Oxford University Press: New York, 2000, pp 33-44). Many of these biologically active glycans contain an essential nine-carbon sugar which is N-acetyl-neuraminic acid (NeuAc). For example, gangliosides are one of the biologically important sialylated glycosphingolipids found in vertebrate cells, nerve cells, as well as in the brain (see Vyas and Schnaar Biochimie 2000, 83, 677-682). In vivo sialylations are catalyzed by a family of enzymes known as sialyltransferases, which contribute to the diversity in the linkage and the chemical structure of sialic acid residues on cell-surface glycoconjugates. Different sialyl-linkages (α-2,3, α-2,6, α-2,8) are elaborated by different sialyltransferases, which share the same donor substrate cytidine monophosphate-sialic acid (CMP-Neu5Ac) but differ in acceptors (see Harduin-Lepers, A. et al. Biochimie 2001, 83, 727-737). Sialyltransferase activity has been shown to correlate with cancer progression and several reasons have been postulated to explain this behavior (see Platt, F. M. et al. Science 1997, 276, 428-431). For example, sialic acids can prevent cell-cell interactions through non-specific charge repulsion effects, which may facilitate metastasis. Secondly, sialyated glycoconjugates can be specifically bound by cell adhesion molecules such as selecting, allowing extravasation of cancer cell (see Feizi, T. Immunol. Rev. 2000, 173, 79-88; and Colin-Hughes, R. Biochimie 2001, 83, 667-676).

Like neuraminidases, the important biological activity of sialyltransferases has prompted chemists to design specific inhibitors of sialyltransferases for elucidating the role of sialyl residues in biological systems (see Wang, X. F. et al. Med. Res. Rev. 2003, 23, 32-47). Several sialyltransferase inhibitors have been developed mostly as CMP-Neu5Ac donor analogues, (see Klohs, W. D. et al. Cancer Res. 1979, 39, 1231-1238; Cambron, L. D. and Leskawa, K. C. Biochem. Biophys. Res. Commun. 1993, 93(2), 585-590; Schaub, C. et al. Glycoconj. J. 1998, 15(4), 345-354; Kijima-Suda, I. et al. Cancer Res. 1986, 46, 858-862; Cohen, S B. and Halcomb, R. L. J. Org. Chem. 2000, 65, 6145-6152; Imamura, M. and Hashimoto, H. Tetrahedron Lett. 1996, 37(9), 1451-1454; Müller, B. et al. Tetrahedron Lett. 1998, 39, 509-512; Amann, F. et al. Chem. Eur. J. 1998, 4(6), 1106-1115; and Compain, P. and Martin. O. V. Bioorg. Med. Chem. 2001, 9, 3077-3092), transition-state analogues, (see Müller, B. et al. Angew. Chem. Int. Ed. 1998, 37(20), 2893-2897; Schwörer, R. and Schmidt, R. R. J. Am. Chem. Soc. 2002, 124, 1632-1637; Schröder, P. N. and Giannis, A. Angew. Chem. Int. Ed. 1999, 38(10), 1379-1380; Sun, H. B. et al. Tetrahedron Lett. 2001, 42, 2451-2453; and Paul, P. et al. J. Biol. Chem. 1993, 268, 12933-12938, and sugar-acceptor analogues (see Kajihara, Y. et al. J. Carbohydr. Chem. 1993, 12(7), 991-996; and Carbohydr. Res. 1993, 247, 179-193).
However, all the known potent sialyltransferase inhibitors are polar and charged. These inhibitors have difficulty exerting their functions in cells or organisms due to low membrane permeability (see Platt, F. M. et al. Science 1997, 276, 428-431). There remains a need for the identification of cell-permeable sialyltransferase inhibitors for in vivo biological study and as pharmaceuticals.
Current sialyltransferase assays use radio-active or fluorescence labeled cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) donor, and fluorescence or ultraviolet (UV)-labeled acceptor (see Paulson, J. C. et al. J. Biol. Chem. 1977, 252, 2363-2371; Gross, H. J. et al. Anal. Biochem. 1990, 186, 127-134; Limberg, G. et al. Liebigs. Ann. 1996, 1773-1784; and Schaub, C. et al. Glycoconj. J. 1998, 15(4), 345-354). These assays require separation of product from donor and acceptor, and are not convenient for the determination of kinetic parameters.
Combinatorial chemistry has developed into a useful method for the rapid identification of lead compounds for drug discovery (see Lam et al. Nature 1991, 354, 82-84; Lam et al. Chem. Rev. 1997, 97(2), 411-448; Pirrung et al. Chem. Rev. 1997, 97(2), 473-488; Nefzi et al. Chem. Rev. 1997, 97(2), 449-472; Young et al. Curr. Opin. Drug Discovery Dev. 2004, 7(3), 318-324). The first step in the process is the facile identification of positive hits from a large collection of compounds. In cases where libraries are prepared in solution, NA activity can be monitored using a synthetic substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid which is cleaved to yield a fluorescent product (4-methylumbelliferone) that can be quantified fluorometrically (see Hochgurtel et al. Proc. Natl. Acad. Sci. USA 2002, 99, 3382-3387; Potier et al. Anal. Biochem. 1979, 94, 287-296). Solution phase assays are not applicable to one-bead-one-compound libraries because it is impossible to identify the bead providing the activity.
Therefore there is a need for the rapid identification of new inhibitors for developing new drugs for the treatment of new and existing strains of flu virus. There also remains a need for a high-throughput screening method for enzyme inhibitors, specifically neuraminidase inhibitors and sialyltransferase inhibitors. The present invention solves this problem by providing an on-bead assay of enzymes, which allows simultaneous monitoring of substrate cleavage and inhibitor efficiency. The substrates and methods for identifying enzyme inhibitors of the present invention can be used in screening of large libraries of compounds for their enzyme inhibitory properties.